666-15 (CREB inhibitor) is a potent and selective CREB inhibitor (IC50: 81 nM).

666-15 is a potent and selective inhibitor of CREB-mediated gene transcription (IC50 = 0.081 ± 0.04 μM). 666-15 also potently inhibited cancer cell growth without harming normal cells [1]. Isoproterenol (ISO) induced cardiac hypertrophy. The rate of ROS production, CREB phosphorylation, and miR-132 expression increased after the addition of ISO. Upregulation of miR-132 was blocked by the specific inhibitor of CREB transcription, 666-15 [2].

In an in vivo MDA-MB-468 xenograft model, 666-15 completely suppressed the tumor growth without overt toxicity [1]. 666-15 was found to be readily bioavailable to achieve pharmacologically relevant concentrations for CREB inhibition. The mice treated with 666-15 showed no evidence of changes in body weight, complete blood count, blood chemistry profile, cardiac contractility and tissue histologies from liver, kidney, and heart [3].

HEK 293T cells in a 10 cm plate were transfected with pCRE-RLuc (6 μg) with Lipofectamine2000 following the manufacturer's instructions. Three hours after transfection, the cells were collected and replated into 96-well plates at ～10?000 cells/well. The cells were allowed to attach to the bottom of the plates overnight. The cells were then treated with different concentrations of different compounds for 30 min when forskolin (10 μM) was added to each well. The cells were incubated for further 5 h before cell lysis using 1× 30 μL Renilla luciferase lysis buffer. An amount of 5 μL of the lysate was combined with 30 μL of benzyl-coelenterazine solution in PBS (pH 7.4, 10 μg/mL). The protein concentration in each well was determined. The Renilla luciferase activity was normalized to protein content in each well and expressed as relative luciferase unit/μg protein (RLU/μg protein). The IC50 was derived from nonlinear regression analysis of the RLU/μg protein–concentration curve [1].

Each 6- to 8-week old BALB/c nude mouse was inoculated subcutaneously at the right flank with MDA-MB-468 cells (5 × 10^6) in 0.1 mL of HBSS with Matrigel (1:1) for tumor development. When the tumor volume reached approximately 100 mm3, the mice were randomized to be treated with either vehicle or 666-15 at 10 mg/kg. 3i was dissolved in 1% N-methylpyrrolidone (NMP), 5% Tween-80 in H2O. The dosing solution was prepared weekly. The mice were treated once a day for 5 consecutive days a week, and the treatment lasted for 5 weeks. During the treatment, the tumor size and body weight were measured 2–3 times a week. The tumors were measured in two dimensions using a digital caliper, and the volume was expressed in mm3 using the formula V = 0.5ab2, where a and b represent the long and short diameters of the tumor, respectively. The tumor volume was normalized to the initial tumor volume at the time of the first treatment [1].